Project Summary: Heart disease is the leading cause of death in the United States. To develop targeted therapeutic approaches for heart and skeletal muscle diseases it is critical to first understand the molecular mechanisms regulating contractile activation and relaxation. We study how different isoforms of myofilament proteins manifest in the different functional properties of the two striated muscle types and how alterations in these proteins lead to changes in function. Progress from this award has clearly demonstrated mechanistic differences in the cooperative activation of cardiac vs. skeletal muscle that suggest different strategies may be needed to improving contraction in impaired tissue. These differences may allow for greater cellular level of controlling force in cardiac muscle, which is required to match stroke volume with venous return on a beat-to-beat basis, i.e. the Frank-Starling Law of the Heart. Additionally, two approaches developed in this award have led to gene delivery-based methods of improving cardiomyocyte function, which is the subject of a new R21 award. In the current proposal we continue to use parallel experiments with cardiac and skeletal systems to study troponin subunit interactions and their role in setting the Ca2+-sensitivity of myofilament contraction, and will look for additional mutants that improve cardiac and skeletal muscle function. Our experiments use a variety of recombinant protein mutants, sophisticated solution biochemical techniques and functional analysis at the level of individual myofilaments, myofibrils cells and tissue. In addition to activation and SL- dependence of contraction, we will now study how troponin subunit interaction properties affect relaxation. This is an understudied but quite important question, in as much as 50% of heart failure may be attributable to diastolic dysfunction. Another new area for the proposal is to study how tropomyosin and myosin isoform composition may influence to coordinated activity of myofilament proteins in determining activation and relaxation kinetics. Finally we will develop a novel 3D-myofilament half- sarcomere model that is 1) spatially explicit and contains the stochastic kinetics of cycling crossbridges and thin filament dynamics and 2) incorporates radial forces associated with actomyosin interaction for more realistic simulation of the SL-dependence of force and understanding of the mechanic-kinetic aspects of relaxation.